Detection of fluorescent signals finds wide application in a variety of situations and under a variety of conditions. Fluorescence has many advantages as a means of generating a detectable signal. Fluorescence does not suffer from the many disadvantages of a radioactive label, while in many cases it provides for a high level of sensitivity. Instrumentation for detection of fluorescence is readily available and fluorescent labels have found application in such diverse situations as immunodiagnostics, detection of nucleic acid bands and protein bands in gel electrophoresis and in fluorescence activated cell sorters. The sensitivity of the fluorescent signal depends upon a number of factors: the possibility of self-quenching; the effect of other molecules associated with the fluorescent molecule on the quantum efficiency of the fluorescence; the effect of the medium on the quantum efficiency and fluorescence characteristics of the fluorescer; the stability of the fluorescer to light; the ability to remove background fluorescence; and the nature of the light source.
For many applications one wishes to have a number of distinguishable fluorescers, so that one can detect different characteristics of a system. For example, in the FACS, there may be an interest in identifying the presence of two characteristics of the cell or other composition. In the hybridization of DNA, one may wish to observe two different DNA sequences, as observed in a gel, on a plate, or the like. Frequently, it is very difficult to obtain fluorescers having emission maxima which are sufficiently different so as to be differentiable while allowing for excitation at the same wave length. There is, therefore, substantial interest in identifying fluorescent markers which permit multiplex determinations by providing for readily differentiable, fluorescent emission maxima, while allowing for excitation with a narrow band radiation source.